A. To determine whether mitochondrial free Ca2+ (Ca2+m) changes upon stimulation of rested guinea pig ventricular myocytes, we utilized our prior observations that the AM form of indo-1 partitions both in the cytosol and in mitochondria. Assuming that indo-1 free acid (FA) localizes only in the cytosol, differences in diastolic indo-1 fluorescence ratio (IFR) upon stimulation, between cells loaded with indo- 1/AM and indo-1/FA, represent non cytosolic, principally Ca2+m accumulation. During the first 35 post rest beats (at 0.5 Hz), the diastolic IFR increases by 32 plus/minus 5% and 5 plus/minus 4% (mean plus/minus SD, n=5) in myocytes loaded with indo-1/AM and FA, respectively, indicating that a significant increase in Ca2+m begins during the initial postrest beats. This result was corroborated by a stimulation-induced increase of the steady IFR in AM loaded cells in which the cytosolic indo-1 fluorescence was quenched with Mn2+. Thus, in guinea pig ventricular myocytes, Ca2+m is rapidly responsive to stimulation- dependent changes in cytosolic Ca2+. B. We determined whether thapsigargin (TG), which inhibits the Ca2+- dependent ATPase (the SR Ca2+ pump) in isolated SR vesicles, depletes the SR in intact rat ventricular myocytes, assessing changes in the SR Ca2+ content as Ca2+i transients elicited rapid, brief pulses of caffeine. After 30 min in 200 nM TG, responses to caffeine were abolished, indicating SR Ca2+ depletion. To determine whether the mode of action of TG is consistent solely with the inhibition of the SR Ca2+ pump, rather than a combined action on the pump and the SR Ca2+ release channel, we compared the effects of TG with that of ryanodine (RY), which inhibits SR function by opening Ca2+ release channels. At low stimulation rates (0.2 Hz) the caffeine-dependent responses are strongly inhibited both in the presence of RY and in TG. Rapid (5 Hz) pacing raises Ca2+i, providing Ca2+ available for the SR uptake. The SR Ca2+ loading occurs and Ca2+ can be released from the SR with caffeine, in the presence of RY but not in TG. Thus, the mode of action of TG on the SR in situ, in intact cardiac myocytes, is consistent with an inhibition of the SR Ca2+ pump.